基于正交试验的人参抗小鼠Lewis肺癌细胞增殖作用的研究
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摘要
目的基于谱效相关分析,探索人参提取物抑制小鼠Lewis肺癌细胞增殖的有效活性成分,为人参用于肺癌治疗的质量控制提供参考依据。方法通过L_9(3~4)正交试验,以提取时间,乙醇浓度,溶剂量为影响因素,对人参超声波提取法进行优化;采用CCK8法检测人参提取物对Lewis细胞增殖的影响,计算IC50;利用超高效液相色谱仪建立不同人参提取物的UPLC指纹图谱;用SPSS22. 0软件的双变量分析法将UPLC指纹图谱与IC_(50)进行相关分析,最后用化学单体进行了活性验证。结果人参体积分数60%的乙醇提取物抑制Lewis细胞增殖的活性强于体积分数90%或体积分数30%的乙醇提取物。不同人参提取物抑制Lewis细胞增殖的IC_(50)范围在0. 708～5. 640 mg·mL~(-1)。谱效相关分析结果表明,人参皂苷Rg1、Re、Rb1、Rb3、Ro、Rh1与抗Lewis细胞增殖活性相关,其中人参皂苷Ro的相关系数高且具有统计学意义(P <0. 05);活性验证结果显示,上述6种单体均有较好的抗Lewis细胞增殖的作用;计算相对活性贡献度可知,人参皂苷Ro是人参体外抗Lewis肺癌细胞增殖的主要活性物质。结论本实验建立的人参提取物抑制Lewis细胞增殖的检测方法对于人参的质量评控具有一定的参考价值,同时初步证明人参皂苷Ro具有明显的抑制Lewis细胞增殖的作用。
OBJECTIVE To explore the anti-proliferation active substance on Lewis lung cancer cells and control quality of Panax ginseng C. A. Mey. for anti-lung cancer,chemical fingerprints and anti-proliferation bioactivity test for spectrum-effect correlation analysis was established in this study. METHODS Using L_9( 3~4) orthogonal test,the extraction time,ethanol concentration,solvent consumption as the factors of ultrasonic extraction of Panax ginseng C. A. Mey. to determine the optimum conditions. CCK8 assay was used to detect the inhibitory effect on proliferation of Lewis lung cancer cells and measure the IC50 values. UPLC was adopted to establish the fingerprint spectra for Panax ginseng C. A. Mey. extracts. Using the double variables of SPSS22. 0 software to correlate the UPLC fingerprint data and IC_(50). We used chemical composition to verify the potency. RESULTS The results showed that 60% aqueous ethanol extract of Panax ginseng C. A. Mey. had more potent anti-proliferation effect than 90% or 30% aqueous ethanol extract. And the IC_(50) of different Panax ginseng C. A. Mey. extracts was 0. 708-5. 640 mg·m L~(-1). Spectrum-effect correlation analysis indicated that ginsenoside Rg1,Re,Rb1,Rb3,Ro and Rh1 had correlation with anti-proliferation bioactivity,and ginsenoside Ro had the highest correlation coefficient( P < 0. 05); furthermore,all of the above six compounds demonstrated anti-proliferation bioactivities. The relative active contributions results suggested that ginsenoside Ro was the main active substance of Panax ginseng C. A. Mey. in the aspect of anti-proliferation of Lewis lung cancer cells. CONCLUSION This study may serve as a reference in the quality control of Panax ginseng C. A. Mey. based on determination of anti-proliferation active substance on Lewis Lung cancer cells. The ginsenoside Ro may be the effective substance.
OBJECTIVE To explore the anti-proliferation active substance on Lewis lung cancer cells and control quality of Panax ginseng C. A. Mey. for anti-lung cancer,chemical fingerprints and anti-proliferation bioactivity test for spectrum-effect correlation analysis was established in this study. METHODS Using L_9( 3~4) orthogonal test,the extraction time,ethanol concentration,solvent consumption as the factors of ultrasonic extraction of Panax ginseng C. A. Mey. to determine the optimum conditions. CCK8 assay was used to detect the inhibitory effect on proliferation of Lewis lung cancer cells and measure the IC50 values. UPLC was adopted to establish the fingerprint spectra for Panax ginseng C. A. Mey. extracts. Using the double variables of SPSS22. 0 software to correlate the UPLC fingerprint data and IC_(50). We used chemical composition to verify the potency. RESULTS The results showed that 60% aqueous ethanol extract of Panax ginseng C. A. Mey. had more potent anti-proliferation effect than 90% or 30% aqueous ethanol extract. And the IC_(50) of different Panax ginseng C. A. Mey. extracts was 0. 708-5. 640 mg·m L~(-1). Spectrum-effect correlation analysis indicated that ginsenoside Rg1,Re,Rb1,Rb3,Ro and Rh1 had correlation with anti-proliferation bioactivity,and ginsenoside Ro had the highest correlation coefficient( P < 0. 05); furthermore,all of the above six compounds demonstrated anti-proliferation bioactivities. The relative active contributions results suggested that ginsenoside Ro was the main active substance of Panax ginseng C. A. Mey. in the aspect of anti-proliferation of Lewis lung cancer cells. CONCLUSION This study may serve as a reference in the quality control of Panax ginseng C. A. Mey. based on determination of anti-proliferation active substance on Lewis Lung cancer cells. The ginsenoside Ro may be the effective substance.
引文
[1] CHEN W Q,SUN K X,ZHENG R S,et al. Report of cancer incidence and mortality in different areas of China,2014[J]. China Cancer(中国肿瘤),2018,27(1):1-14.
[2] LUO L M,SHI Y M,JANG Y N,et al. Advance in commponents with antitumor effect of Panax ginseng and their mechanisms[J]. Chin Tradit Herb Drugs(中草药),2017,48(3):582-596.
[3] LEEM D G,SHIN J S,KIM K T,et al. Dammarane-type triterpene ginsenoside-Rg18 inhibits human non-small cell lung cancer A549 cell proliferation via G1 phase arrest[J]. Oncol Lett,2018,15(4):6043-6049.
[4] BI L,YAN X,YANG Y,et al. The component formula of Salvia miltiorrhiza and Panax ginseng induces apoptosis and inhibits cell invasion and migration through targeting PTEN in lung cancer cells[J]. Oncotarget,2017,8(60):101599-101613.
[5] TENG S,WANG Y,LI P,et al. Effects of R type and S type ginsenoside Rg3 on DNA methylation in human hepatocarcinoma cells[J]. Mol Med Rep,2017,15(4):2029-2038.
[6] YUAN Z,JIANG H,ZHU X J,et al. Ginsenoside Rg3 promotes cytotoxicity of paclitaxel through inhibiting NF-kappaB signaling and regulating Bax/Bcl-2 expression on triple-negative breast cancer[J]. Biomed Pharmacother,2017,89:227-232.
[7] ZHOU Y,ZHENG X,LU J,et al. Ginsenoside 20(S)-Rg3 inhibits the warburg effect via modulating DNMT3A/MiR-532-3p/HK2 pathway in ovarian cancer cells[J]. Cell Physiol Biochem,2018,45(6):2548-2559.
[8] WU Q,DENG J,FAN D,et al. Ginsenoside Rh4 induces apoptosis and autophagic cell death through activation of the ROS/JNK/p53 pathway in colorectal cancer cells[J]. Biochem Pharmacol,2018,148:64-74.
[9] YAO M,LV J M,ZHANG Q,et al. Study on chemical constituents and pharmacological effects of ginseng[J]. Jilin J Tradit Chin Med(吉林中医药),2017,37(12):1261-1263.
[10] MAJEED F,MALIK F Z,AHMED Z,et al. Ginseng phytochemicals as therapeutics in oncology:recent perspectives[J].Biomed Pharmacother,2018,100:52-63.
[11] DAI D,ZHANG C F,WILLIAMS S,et al. Ginseng on cancer:potential role in modulating inflammation-mediated angiogenesis[J]. Am J Chin Med,2017,45(1):13-22.
[12] LEE J H,KWON K R,CHO C K,et al. Advanced cancer cases treated with cultivated wild ginseng phamacopuncture[J]. J Acupunct Meridian Stud,2010,3(2):119-124.
[13] LIU C,GONG Q,CHEN T,et al. Treatment with 20(S)-ginsenoside Rg3 reverses multidrug resistance in A549/DDP xenograft tumors[J]. Oncol Lett,2018,15(4):4376-4382.
[14] WANG Y,XU H,LU Z,et al. Pseudo-ginsenoside Rh2 induces A549 cells apoptosis via the Ras/Raf/ERK/p53 pathway[J].Exp Ther Med,2018,15(6):4916-4924.
[15] GE G,YAN Y,CAI H. Ginsenoside Rh2 inhibited proliferation by inducing ROS mediated ER stress dependent apoptosis in lung cancer cells[J]. Biol Pharm Bull,2017,40(12):2117-2124.
[16] DUAN Z,DENG J,DONG Y,et al. Anticancer effects of ginsenoside Rk3 on non-small cell lung cancer cells:in vitro and in vivo[J]. Food Funct,2017,8(10):3723-3736.
[17] HE Q,TU C,WANG J B,et al. Antiplatelet aggregation bioactivity of Polygni Multiflori Radix with chemical fingerprints and spectrum-effect correlation analysis[J]. China J Chin Mater Med(中国中药杂志),2017,42(9):1679-1684.
[18] WANG Z L,OUYANG L F,ZHANG Q,et al. A progress on the extraction and purification of Ginseng total saponins[J]. World Sci Technol Mod Tradit Chin Med(世界科学技术-中医药现代化),2016,18(9):1596-1601.
[19] XIN X J,CHEN Y,CONG J H. Effects of different extraction methods on the yield of ginsenosides[J]. Forest by-Product Special China(中国林副特产),2015,(1):34-35.
[20] DONG W,GAO Y G,HE Z M,et al. Research progress in extraction and separation technology of ginsenosides[J]. Sci Technol Food Ind(食品工业科技),2014,35(17):366-369.
[21] Ch. P(2015)VolⅠ(中国药典2015年版.一部)[S].2015:8.
[22] LIU C X,CHEN S L,XIAO X H,et al. A new concept on quality marker of Chinese material medica:quality control for Chinese medicine products[J]. Chin Tradit Herb Drugs(中草药),2016,47(9):1443-1457.
[23] WANG X L,LIU X,HAN Y Q,et al. Overview on main research method of effective material basis of Chinese materia medica[J]. Chin Tradit Herb Drugs(中草药),2018,49(4):941-947.
[24] ZHAO X M,PU S B,ZHAO Q G,et al. Preliminary study on effective components of Tripterygium wilfordii for liver toxicity based on spectrum-effect correlation analysis[J]. China J Chin Mater Med(中国中药杂志),2016,41(15):2915-2921.
[25] SHEN C H,LIU C T,SONG X J,et al. Evaluation of analgesic and anti-inflammatory activities of Rubia cordifolia L. by spectrum-effect relationships[J]. J Chromatogr B Analyt Technol Biomed Life Sci,2018,1090:73-80.
[26] XIAO X H,WANG J B,YAN D. Studies and a pplication of biological evaluationin the quality standardization of Chinese medicines[J]. World Sci Technol Mod Tradit Chin Med(世界科学技术-中医药现代化),2014,16(3):514-518.
[27] QIN S,XU X X,LI Q,et al. Ginsenoside Ro pharmacological effects review[J]. Nat Product Res Develop(天然产物研究与开发),2014,26(S2):346-348.
[28] ZHENG K,LI Y,WANG S,et al. Inhibition of autophagosomelysosome fusion by ginsenoside Ro via the ESR2-NCF1-ROS pathway sensitizes esophageal cancer cells to 5-fluorouracil-induced cell death via the CHEK1-mediated DNA damage checkpoint[J]. Autophagy,2016,12(9):1593-1613.
[29] JIANG Z,QIAN J,DONG H,et al. The traditional Chinese medicine achyranthes bidentata and our de novo conception of its metastatic chemoprevention:from phytochemistry to pharmacology[J]. Sci Rep,2017,7(1):3888.
[30] LI Z W. The fuction and mechanism of hinsenoside Ro mediated ATAT5 pathway in promoting the leukemia-derived dendritic differentiation[D]. Jinan:University of Jinan,2017.
[31] WANG X P,WANG J,BAI J Q,et al. The antitumor effects of ginsenoside Ro on proliferaton and apoptosis of gastric cancer cells MFC[J]. West China J Pharm Sci(陕西中医药大学学报),2017,40(4):110-113.
[32] ZHENG S W. Effect of Panax ginseng and Ginsenosides on Melanoma and Angiogenesis[D]. Changchun:Evaluation and Utilization of Chinese Medicinal Materials,2016.
[2] LUO L M,SHI Y M,JANG Y N,et al. Advance in commponents with antitumor effect of Panax ginseng and their mechanisms[J]. Chin Tradit Herb Drugs(中草药),2017,48(3):582-596.
[3] LEEM D G,SHIN J S,KIM K T,et al. Dammarane-type triterpene ginsenoside-Rg18 inhibits human non-small cell lung cancer A549 cell proliferation via G1 phase arrest[J]. Oncol Lett,2018,15(4):6043-6049.
[4] BI L,YAN X,YANG Y,et al. The component formula of Salvia miltiorrhiza and Panax ginseng induces apoptosis and inhibits cell invasion and migration through targeting PTEN in lung cancer cells[J]. Oncotarget,2017,8(60):101599-101613.
[5] TENG S,WANG Y,LI P,et al. Effects of R type and S type ginsenoside Rg3 on DNA methylation in human hepatocarcinoma cells[J]. Mol Med Rep,2017,15(4):2029-2038.
[6] YUAN Z,JIANG H,ZHU X J,et al. Ginsenoside Rg3 promotes cytotoxicity of paclitaxel through inhibiting NF-kappaB signaling and regulating Bax/Bcl-2 expression on triple-negative breast cancer[J]. Biomed Pharmacother,2017,89:227-232.
[7] ZHOU Y,ZHENG X,LU J,et al. Ginsenoside 20(S)-Rg3 inhibits the warburg effect via modulating DNMT3A/MiR-532-3p/HK2 pathway in ovarian cancer cells[J]. Cell Physiol Biochem,2018,45(6):2548-2559.
[8] WU Q,DENG J,FAN D,et al. Ginsenoside Rh4 induces apoptosis and autophagic cell death through activation of the ROS/JNK/p53 pathway in colorectal cancer cells[J]. Biochem Pharmacol,2018,148:64-74.
[9] YAO M,LV J M,ZHANG Q,et al. Study on chemical constituents and pharmacological effects of ginseng[J]. Jilin J Tradit Chin Med(吉林中医药),2017,37(12):1261-1263.
[10] MAJEED F,MALIK F Z,AHMED Z,et al. Ginseng phytochemicals as therapeutics in oncology:recent perspectives[J].Biomed Pharmacother,2018,100:52-63.
[11] DAI D,ZHANG C F,WILLIAMS S,et al. Ginseng on cancer:potential role in modulating inflammation-mediated angiogenesis[J]. Am J Chin Med,2017,45(1):13-22.
[12] LEE J H,KWON K R,CHO C K,et al. Advanced cancer cases treated with cultivated wild ginseng phamacopuncture[J]. J Acupunct Meridian Stud,2010,3(2):119-124.
[13] LIU C,GONG Q,CHEN T,et al. Treatment with 20(S)-ginsenoside Rg3 reverses multidrug resistance in A549/DDP xenograft tumors[J]. Oncol Lett,2018,15(4):4376-4382.
[14] WANG Y,XU H,LU Z,et al. Pseudo-ginsenoside Rh2 induces A549 cells apoptosis via the Ras/Raf/ERK/p53 pathway[J].Exp Ther Med,2018,15(6):4916-4924.
[15] GE G,YAN Y,CAI H. Ginsenoside Rh2 inhibited proliferation by inducing ROS mediated ER stress dependent apoptosis in lung cancer cells[J]. Biol Pharm Bull,2017,40(12):2117-2124.
[16] DUAN Z,DENG J,DONG Y,et al. Anticancer effects of ginsenoside Rk3 on non-small cell lung cancer cells:in vitro and in vivo[J]. Food Funct,2017,8(10):3723-3736.
[17] HE Q,TU C,WANG J B,et al. Antiplatelet aggregation bioactivity of Polygni Multiflori Radix with chemical fingerprints and spectrum-effect correlation analysis[J]. China J Chin Mater Med(中国中药杂志),2017,42(9):1679-1684.
[18] WANG Z L,OUYANG L F,ZHANG Q,et al. A progress on the extraction and purification of Ginseng total saponins[J]. World Sci Technol Mod Tradit Chin Med(世界科学技术-中医药现代化),2016,18(9):1596-1601.
[19] XIN X J,CHEN Y,CONG J H. Effects of different extraction methods on the yield of ginsenosides[J]. Forest by-Product Special China(中国林副特产),2015,(1):34-35.
[20] DONG W,GAO Y G,HE Z M,et al. Research progress in extraction and separation technology of ginsenosides[J]. Sci Technol Food Ind(食品工业科技),2014,35(17):366-369.
[21] Ch. P(2015)VolⅠ(中国药典2015年版.一部)[S].2015:8.
[22] LIU C X,CHEN S L,XIAO X H,et al. A new concept on quality marker of Chinese material medica:quality control for Chinese medicine products[J]. Chin Tradit Herb Drugs(中草药),2016,47(9):1443-1457.
[23] WANG X L,LIU X,HAN Y Q,et al. Overview on main research method of effective material basis of Chinese materia medica[J]. Chin Tradit Herb Drugs(中草药),2018,49(4):941-947.
[24] ZHAO X M,PU S B,ZHAO Q G,et al. Preliminary study on effective components of Tripterygium wilfordii for liver toxicity based on spectrum-effect correlation analysis[J]. China J Chin Mater Med(中国中药杂志),2016,41(15):2915-2921.
[25] SHEN C H,LIU C T,SONG X J,et al. Evaluation of analgesic and anti-inflammatory activities of Rubia cordifolia L. by spectrum-effect relationships[J]. J Chromatogr B Analyt Technol Biomed Life Sci,2018,1090:73-80.
[26] XIAO X H,WANG J B,YAN D. Studies and a pplication of biological evaluationin the quality standardization of Chinese medicines[J]. World Sci Technol Mod Tradit Chin Med(世界科学技术-中医药现代化),2014,16(3):514-518.
[27] QIN S,XU X X,LI Q,et al. Ginsenoside Ro pharmacological effects review[J]. Nat Product Res Develop(天然产物研究与开发),2014,26(S2):346-348.
[28] ZHENG K,LI Y,WANG S,et al. Inhibition of autophagosomelysosome fusion by ginsenoside Ro via the ESR2-NCF1-ROS pathway sensitizes esophageal cancer cells to 5-fluorouracil-induced cell death via the CHEK1-mediated DNA damage checkpoint[J]. Autophagy,2016,12(9):1593-1613.
[29] JIANG Z,QIAN J,DONG H,et al. The traditional Chinese medicine achyranthes bidentata and our de novo conception of its metastatic chemoprevention:from phytochemistry to pharmacology[J]. Sci Rep,2017,7(1):3888.
[30] LI Z W. The fuction and mechanism of hinsenoside Ro mediated ATAT5 pathway in promoting the leukemia-derived dendritic differentiation[D]. Jinan:University of Jinan,2017.
[31] WANG X P,WANG J,BAI J Q,et al. The antitumor effects of ginsenoside Ro on proliferaton and apoptosis of gastric cancer cells MFC[J]. West China J Pharm Sci(陕西中医药大学学报),2017,40(4):110-113.
[32] ZHENG S W. Effect of Panax ginseng and Ginsenosides on Melanoma and Angiogenesis[D]. Changchun:Evaluation and Utilization of Chinese Medicinal Materials,2016.